Enhancer of zeste homolog 2 (EZH2) catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) and elevated levels of EZH2 as well as mutations in the Ezh2 gene have been linked to cancer progression. While EZH2 concentrations are frequently elevated in solid tumors loss of function mutations have been identified in some hematopoietic disorders. At this point it is not clear whether EZH2 conveys tumor promoting or suppressing functions. Mouse genetics and tissue specimen of human breast cancer were employed to address the role of EZH2 in mammary tissue and breast cancer. Studies performed in this reporting period demonstrated that the methyltransferase EZH2 is not required for mammary cancer development, although high EZH2 and low H3K27me3 correlate with poor prognosis of ER-positive breast cancers (Bae et al., 2014). Levels of H3K27me3 and EZH2 and their prognostic significance were evaluated in one hundred forty six cases of breast cancer. H3K27me3 levels were higher in HER2-negative samples. EZH2 expression was higher in cancers that were LN+, size > 20mm, and with higher tumor grade and stage. H3K27me3 levels and EZH2 expression were identified as independent prognostic factors for overall survival for all the breast cancers studied as well as the ER-positive subgroup. The combination of low H3K27me3 and high EZH2 expression levels were significantly associated with shorter survival. Using a mouse mammary cancer model based on the loss of Brca1 it was determined that the presence of EZH2 was not required for the establishment and progression of mammary cancers. Moreover, cancer cells that lacked both Brca1 and Ezh2 still exhibited the presence of genomic H3K27me3. This demonstrated that EZH2 is not a mandatory H3K27 methyltransferase in mammary neoplasia and provided genetic evidence for biological independence between H3K27me3 and EZH2 in this tissue. During this reporting period we have continued to explore the role of the histone methyltransferases EZH1 and EZH2 in the biology of mammary epithelium. We have determined that neither EZH1 nor EZH2 are required for mammary development, suggesting that these enzymes can compensate for each other. This is supported by the observation that H3K27me3 levels are overtly normal in the absence of either methyltransferase. However, EZH2 controls the temporal progression of mammary differentiation during pregnancy. Precocious activation of genetic programs in the absence of EZH2 is likely mediated by the unscheduled interaction of the transcription factor STAT5 with the mammary genome.